Cold-sensitive expression of transformation by a host range mutant of type 5 adenovirus.
Cold-sensitive expression of transformation by a host range mutant of type 5 adenovirus.
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5 型腺病毒宿主范围突变体的冷敏感转化表达。
DOI：
10.1073/pnas.80.5.1352
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发表时间：
1983
影响因子：
11.1
通讯作者：
Fisher,PB
中科院分区：
文献类型：
--
作者：
Babiss,LE;Ginsberg,HS;Fisher,PB
hr1是5型腺病毒（Ad5）的E1a（0-4.5个图谱单位）宿主范围突变体，当细胞在37℃培养时，hr1转化克隆大鼠胚胎成纤维细胞（CREF）细胞系的频率比野生型（wt） Ad5高5倍。然而，如果细胞感染hr1并保持在32℃，则不会发生形态转化。当感染细胞在感染后2周从32℃转移到37℃时，6周的转化频率与在37℃连续生长的细胞相同，而在感染后2周从37℃转移到32℃的培养显示形态转化减少96%以上。hr1-transformed细胞有一个成纤维细胞的形态与wt Ad5-transformed细胞典型的上皮样的形态,但hr1上——和wt Ad5-transformed细胞有类似的饱和密度,增长速度,和琼脂克隆效率化验时37度C。然而,当细胞改变了hr1上在37摄氏度生长在32摄氏度,他们有一个饱和密度接近正常CREF细胞和琼脂的效率增长低于wt-transformed细胞。对2个hr1转化的克隆细胞系A2和B3的DNA转移/杂交分析表明，A2细胞包含Ad5基因组的完整整合拷贝，而B3细胞仅整合了Ad5基因组的一部分。RNA转移和RNA/DNA过滤杂交分析表明，当A2和B3细胞系在32℃和37℃下生长时，病毒信使RNA的类型和转录的相对RNA量相似。间接免疫荧光与携带ad诱导肿瘤的仓鼠的抗血清显示染色温度依赖性-即。,细胞生长在37摄氏度或从32度到37摄氏度包含强烈,核地区颗粒染色,而染色显著降低在细胞培养在32摄氏度,细胞从37摄氏度转向32度C。这些研究结果表明,基因产物的影响hr1上突变冷敏感和对的表达至关重要的特征识别转化细胞。
hr1, an E1a (0-4.5 map units) host range mutant of type 5 adenovirus (Ad5), transformed a cloned rat embryo fibroblast (CREF) cell line at about a 5-fold higher frequency than wild-type (wt) Ad5 when cells were cultured at 37 degrees C. However, if the cells were infected with hr1 and maintained at 32 degrees C morphological transformation did not occur. When infected cells were shifted from 32 degrees C to 37 degrees C 2 wk postinfection, the frequency of transformation by 6 wk was identical to that of cells grown continuously at 37 degrees C, whereas cultures shifted from 37 degrees C to 32 degrees C 2 wk postinfection displayed a greater than 96% reduction in morphological transformation. hr1-transformed cells had a fibroblastic morphology as contrasted with the typical epithelioid morphology of wt Ad5-transformed cells, but hr1- and wt Ad5-transformed cells had similar saturation densities, growth rates, and agar cloning efficiencies when assayed at 37 degrees C. However, when cells transformed by hr1 at 37 degrees C were grown at 32 degrees C, they had a saturation density close to that of normal CREF cells and grew at a lower efficiency in agar than wt-transformed cells. DNA transfer/hybridization analysis of two hr1-transformed cloned cell lines, A2 and B3, indicated that A2 cells contained a complete integrated copy of the Ad5 genome, whereas in B3 cells only part of the Ad5 genome was integrated. RNA transfer and RNA/DNA filter hybridization analyses indicated that the types of viral messenger RNAs and the relative amounts of RNA transcribed were similar in the A2 and B3 cell lines when they were grown at 32 degrees C and 37 degrees C. Indirect immunofluorescence, with antisera from hamsters bearing Ad-induced tumors, indicated a temperature dependence in staining--i.e., cells grown at 37 degrees C or shifted from 32 degrees C to 37 degrees C contained intense, particulate staining in the nuclear region, whereas the staining was decreased significantly in cells cultured at 32 degrees C and in cells shifted from 37 degrees C to 32 degrees C. These findings indicate that the gene product affected by the hr1 mutation is cold sensitive and is essential for the expression of the characteristics that identify the transformed cell.